Method for inhibition or treatment of colon tumorigenesis

ABSTRACT

A method is provided for inhibiting the development of colon tumorigenesis in a mammal by administering to the mammal a pharmacologically effective amount of an isothiocyanate selected from the group consisting of sulforaphane and phenethyl isothiocyanate.

FIELD OF INVENTION

This invention relates to treatment for the inhibition or reduction ofcolon tumorigenesis in a mammal by the administration of apharmaceutical compound.

BACKGROUND OF INVENTION

Evidence from epidemiological studies suggests an association ofconsumption of cruciferous vegetables and a reduced risk of colon cancer(31). Although a number of compounds in these vegetables maycollectively cause the beneficial effects, there has been a need toidentify the exact role of each compound that contributes to theprotective effects. Ample data from laboratory animal studies have shownthat isothiocyanates (ITCs), one of the major constituents ofcruciferous vegetables, are promising chemopreventive agents againstcancers at various sites, including lung, esophagus, liver, mammary, andbladder (3,32,33). These laboratory results support a potential role ofdietary ITCs in reducing risk of certain human cancers. However, it iswell known to those of skill in the art that chemopreventive agentsaffect different tissues differently.

Previously, for example, it has been reported that while phenylhexylITC, a synthetic homologue of PEITC, is a potent inhibitor of lungtumorigenesis, it enhanced colon and esophagus tumorigenesis in rats,possibly due to its tissue cytotoxicity at the dose level studied(29,30). These results illustrate the importance of considering tissuespecificity and of choosing the appropriate dose range for specifictissues in chemoprevention studies.

A recent case-control study reported that broccoli consumption is linkedto a lowered risk of colon cancer, and the protective effect isespecially evident in individuals with a glutathione transferase (GST)M1 null genotype (1). Because GSTs facilitate the conjugation of ITCsresulting in their excretion as the N-acetylcysteine (NAC) conjugatesvia the mercapturic acid pathway, it has been suggested that the ITCcompounds in broccoli may play a role in the protection of human coloncancer (2). Sulforaphane (SFN) is the predominant ITC found in broccoliwhich has been studied for its chemopreventive potential due to itsactivity in the induction of phase II enzymes involved in carcinogendetoxification and elimination (3). However, so far no animal data isavailable regarding the effects of SFN on colon tumorigenesis. Severallaboratory animal studies have shown that phenethyl ITC (PEITC), aprincipal constituent in watercress, is a potent chemopreventive agentfor cancers of the breast, lung, and esophagus (4,5,6). However, thereis insufficient data for PEITC on colon cancer.

Considering the natural abundance of SFN and PEITC in broccoli andwatercress, respectively, and their potential as chemopreventive agents,it is surprising that little is known about the effects of these agentson colon tumorigenesis. A lower homologue of PEITC, benzyl ITC (BITC)has been shown to inhibit colon tumor incidence in AOM treated ratsduring the initiation phase, but not during the post-initiation phase(34). Another short-term study, however, has reported that both PEITCand BITC given in the diet at similar dose levels were inactive towardsACF formation, in fact, BITC was found to slightly induce ACF formation(10). By contrast, the present inventors have now demonstrated for thefirst time that both PEITC and SFN inhibit colonic, ACF, independent ofwhether they are administered before or after carcinogen exposure.

SUMMARY OF THE INVENTION

There is provided in accordance with one embodiment of the invention amethod for inhibiting tumor development in a mammal. The methodcomprises administering to the mammal a pharmacologically effectiveamount of an isothiocyanate selected from the group consisting ofsulforaphane and phenethyl isothiocyanate. The sulforaphane may beisolated from broccoli and the phenethyl isothiocyanate may be isolatedfrom watercress. The isothiocyanate is preferably administered to themammal as a purified compound, either alone or in a composition with apharmacologically acceptable carrier, excipient or diluent or with abeverage or foodstuff.

In a preferred embodiment of the invention, the mammal is a human andthe isothiocyanate is administered to the human as a dietary supplement.In a preferred treatment regimen, the isothiocyanate is administered tothe human in a dosage of between about 0.5 and 12 mg/kg body weight perday.

In another embodiment of the invention there is provided a method fortreating colon tumor formation in a mammal in need of such treatment.The method comprises administering to the mammal a pharmacologicallyeffective amount of an isothiocyanate selected from the group consistingof sulforaphane and phenethyl isothiocyanate.

The above and other features and advantages of the invention will befound in the detailed description which follows below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the body weight over time of rats treated withisothiocyanates in accordance with the invention before (initiation) AOMtreatment and rats in a control group; and

FIG. 2 is a graph showing the body weight over time of rats treated withisothiocyanates in accordance with the invention after (post-initiation)AOM treatment and rats in control group.

DETAILED DESCRIPTION

As used herein, the abbreviations have the following meanings: ITCs,isothiocyanates; SFN, sulforaphane; PEITC, phenethyl isothiocyanate;BITC, benzyl isothiocyanate; AFC, aberrant crypt foci; NAC,N-acetylcysteine; AOM, azoxymethane; NNK,-(4-methylnitrosamino)-1-(3-pyridyl)-2-butanone; CYP, cytochrome P450;NDMA, N,N-dimethylnitrosamine. All references cited herein are herebyspecifically incorporated into this disclosure by reference.

The inventors designed a bioassay to examine whether SFN and PEITC caninhibit the formation of aberrant crypt foci (ACF) induced byazoxymethane (AOM) in Fischer rats. ACF has been recognized as an earlypreneoplastic lesion of colon cancer (7-9) and it is generally observedthat agents that inhibit colonic ACF formation would showchemopreventive activity against colon cancer (10). The inventors alsoincluded in this study the NAC conjugates of SFN and PEITC since theirprevious studies showed that certain ITC-NAC conjugates are promisingchemopreventive agents for lung tumorigenesis, probably by a gradualrelease of the parent ITCs via a dissociation reaction (11,12). Thestructures of SFN and PEITC and their NAC conjugates are shown below.Two treatment regimens in which SFN and PEITC or their NAC conjugateswere administered either after (post-initiation) or before (initiation)AOM treatment were used in the bioassay in order better to define thepossible mechanism of action.

Male F344 rats were from Charles River (Kingston, N.Y.). They were fedAIN-76A diet (5% corn oil) and tap water ad libitum. Animals weremaintained under standard conditions (12-h light/12-h dark cycle, 50%relative humidity at 21° C.). At six-weeks of age, they were randomlydivided into six per groups as shown in Table 1, except for the controlgroups 10 to 14, which consist of three animals. PEITC and SFN werepurchased from Aldrich Chemical Company (Milwaukee, Wis.) and LTK Labs,Inc. (St. Paul, Minn.), with purity >99% and >97%, respectively. Thecorresponding NAC conjugates were prepared by a published method(13,26). The purity of the conjugates was greater than 97% as determinedby high-performance liquid chromatography. AOM obtained from Ash-Stevens(Detroit, Mich.) was dissolved in saline and administered subcutaneouslyonce a week for two weeks. SFN and PEITC in corn oil and the NACconjugates in saline (10% DMSO) were given by gavage. Groups 2 to 5 weregiven 5 μmol SFN or PEITC or 20 μmol of the NAC conjugates, three doseseach week for eight weeks, beginning two days after the last dose ofAOM. Groups 6 to 9 were treated with three doses of ITC compounds (20μmol ITC or 50 μmol ITC-NAC) once daily and the last dose was given 2 hrbefore AOM dosing. This dosing regimen was repeated during the secondweek of AOM dosing. Groups 10 to 13 were treated with ITCs (5 μmol/dose)or the conjugates (20 μmol/dose) only, three times weekly for eightweeks. Group 14 served as the control group. The bioassay was terminatedat week 10 after the second AOM treatment. The colon was processed formicroscopic examination and the ACF were recorded using a standardprocedure described previously (14). ACF were distinguished from thesurrounding normal crypts by their increased size, significantlyincreased distance from lamina to basal surface of cells, and the easilydiscernable pericryptal zone. For statistical analysis, means werecompared among the groups using one-way analysis of variance (ANOVA)followed by Fisher's protected t-test.

No significant differences in body weights were seen in any of thetreated groups compared with the control group, indicating that doses ofITCs and the conjugates used did not cause overt toxicity (see FIGS. 1and 2). This bioassay showed that post-treatment by oral administrationof SFN and PEITC at 5 μmol three doses weekly for eight weeks and theirNAC conjugates at 20 μmol by the same regimen inhibited the formation ofACF. These treatments reduced the total ACF from 153 to 10-1016 (p<0.01)and multicrypt foci (>4 crypts/focus) from 52 to 27-37 (p<0.04) as shownin Table 1 (Groups 1 to 5). Since the ITC conjugates are presumably lesstoxic than the parent ITCs, the doses of the conjugates were 4 timesthat of SFN and PEITC (15,16). However, the inventors did not findsignificant difference in the inhibition of ACF between the ITCs andtheir NAC conjugates, suggesting the conjugates themselves are not asactive. Similar dose-effect relationships were observed previouslybetween parent ITCs and their conjugates towards the inhibition of lungtumorigenesis (7). These results, together with those from theinventors' earlier studies (17,18), suggest that ITC conjugates rendertheir inhibitory activity in part by the deconjugation to the parentITCs. Although the mechanism of inhibition of ACF at the post-initiationphase is not currently clear, recent studies have shown that PEITC andrelated ITCs induce p53-dependent or c-jun kinase-mediated apoptosis incultured cells (19-21). In addition, NAC produced by deconjugation is aknown antioxidant which has been recently shown to inhibit mousefibroblast cell proliferation by blocking cell in G1 phase (22). Allthese activities could have contributed to the inhibition of ACFformation during the post-initiation of ACF formation.

Studies by the inventors and others showed that pretreatment of animalswith PEITC and other related ITCs blocked chemical-inducedtumorigenicity by inhibiting cytochrome p450 (CYP) enzymes responsiblefor the activation of carcinogens, and consequently reducing DNA damage(23-25). The inventors investigated the effects of pretreatment with SFNand PEITC on the AOM-induced colonic ACF formation in Fischer rats. Theresults showed that pretreatment with SFN or PEITC significantlydecreased total number of ACF from 153 to 109 (0<0.004) or 115 (p<0.01),respectively, and multicrypt foci (>4 crypts) from 52 to 35 (p<0.03) forboth compounds (Table 1 below). The inventors have previously shown thatPEITC and PEITC-NAC are inhibitors of N,N-dimethylnitrosamine (NDMA)demethylase (CYP2E1) in rat liver microsomes (26). Like NDMA, AOM ismetabolically activated by CYP2E1 to methyazoxymethanol which can yielda DNA methylating species (27). Thus, inhibition of CYP2E1 by theseagents in the rat liver may constitute an important mechanism for theinhibition, since CYP enzyme activity in colonic tissue is low comparedto that in liver (24). SFN-NAC also reduced total AFC to 120 (p<0.02),however, it had no significant effect on multicrypt foci (>4crypts/focus, 15% inhibition). PEITC-NAC, on the other hand, appeared toenhance ACF formation (total number of ACF is 198, p<0.002; number ofmulticrypt foci is 74, p<0.008) (Table 1). The adverse effect caused byPEITC-NAC is unexpected in view of the inhibition by its parent ITC. Atpresent, it is not clear as to why there is such a sharp contrasttowards ACF formation for PEITC-NAC compared to other ITC compoundsstudied here. TABLE 1 Effects of SFN and PEITC on the formation ofaberrant crypt foci induced by AOM Average Body Weight Dose of ITC atNumber of Compounds Ter- aberrant crypt foci Treatment Group^(a) (μmol)mination >4 Crypts Total  1. AOM — 310 52 153  2. AOM→SFN 5 301 30(42)^(b,c) 103 (33)^(d)  3. AOM→SFN-NAC 20 297 31 (40)^(c) 116 (24)^(c) 4. AOM→PEITC 5 306 27 (48)^(c) 100 (35)^(d)  5. AOM→PEITC- 20 313 38(27)^(f) 113 (26)^(e)    NAC  6. SFN→AOM 20 310 35 (33)^(f) 109 (29)^(c) 7. SFN-NAC→AOM 50 304 44 (15) 120 (22)^(c)  8. PEITC→AOM 20 307 35(33)^(f) 115 (25)^(c)  9. PEITC-NAC→ 50 303 74 (−42)^(c) 198 (−29)^(e)   AOM 10. SFN 5 309  0  0 11. SFN-NAC 20 312  0  0 12. PEITC 5 333  0 0 13. PEITC-NAC 20 301  0  0 14. Control — 320  0  0^(a)In Groups 2 to 5, ITC compounds are administered duringpost-initiation phase and in Groups 6 to 9 ITC compounds were givenduring initiation phase.^(b)Percent of inhibition compared to Group 1.^(c)Significantly different from Group 1 at p < 0.01.^(d)Significantly different from Group 1 at p < 0.0001^(e)Significantly different from Group 1 at p < 0.001^(f)Significantly different from Group 1 at p < 0.05Preferred Dose Levels and Toxicity Considerations

In the above study a colon carcinogen, AOM, was used to initiateprecancerous lesions in the colon; the dose was considerably higher thanthe anticipated dose of any carcinogen to which humans would beinadvertently subjected. The chemopreventive agents were administered intwo types of treatments. In the first type of treatment, rats were dosedafter AOM was given with 5 μmole of either sulforaphane (SFN) [6.3mg/kg] or phenethyl isothiocyanate (PEITC) [5.8 mg/kg] or 20 μmole ofeither the N-acetylcysteine conjugate of SFA (SFN-NAC) [48.6 mg/kg] orthe N-acetylcysteine conjugate of PEITC (PEITC-NAC) [46.6 mg/kg] threetimes per week for eight weeks. In the second treatment regimen, therats received 20 μmole of the respective ITCs or 50 μmole of theN-acetylcysteine conjugates four days per week for two weeks prior toAOM administration once each week. Symptoms of gastrointestinal toxicityor irritation (bloody mucus in feces) were observed very early in thesecond treatment regimen, suggesting that the dose levels used may havebeen in excess of a dose safe for human consumption.

Additional toxicity information regarding PEITC is available from a 90day study in rats, in which 500, 1500, and 2500 ppm was administered inthe diet (35). Dietary PEITC reduced weight gain in male rats at thehigh dose level by approximately 9.5% (not significant). In addition,liver weights of male rats ingesting 1500 and 2500 ppm PEITC weresignificantly elevated. Changes in the epithelial lining of theforestomach of both male and female rats at 2500 ppm were also observed.In another study with Beagle dogs, PEITC was administered daily ingelatin capsules. Diarrhea and vomiting, with gastrointestinalirritation, occurred sporadically at dose levels of 2 mg/kg and higher.Signs of irritation of the mucosa of the urinary bladder were reportedat 4 mg/kg/day and higher. Dose related weight loss occurred in bothmale and female dogs, which became statistically significant in femalesat 8 mg/kg. No other signs or symptoms of toxicity were reported (36).

In a preliminary study in humans presently being conducted to determinethe efficacy of PEITC as a chemopreventive agent for lung cancer insmokers, volunteers have been given 40 mg PEITC (approx. 0.6 mg/kg) ingelatin capsules. No adverse effects from this dose have been observedafter repeated dosing every four hours.

On the basis of existing toxicity information and efficacy data, theinventors suggest the following safe dose ranges for human consumption:PEITC 0.6 to 1.2 mg/kg body weight PEITC-NAC 6.0 to 12 mg/kg body weightSFN 0.55 to 1.1 mg/kg body weight SFN-NAC 5.5 to 11 mg/kg body weight

The components of the present invention may be administered as a dietarysupplement. For example, they may be administered orally, either aloneor with water or another beverage or with a foodstuff.

There may be many modifications and variations of the methods andcompounds set forth hereinabove. These modifications and variations willnot depart from the scope of the invention, if defined by the followingclaims and equivalents thereof.

REFERENCES

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1-20. (canceled)
 21. A method for inhibiting colon tumor development ina mammal, said method comprising administering to the mammal apharmacologically effective amount of an isothiocyanate selected fromthe group consisting of sulforaphane, phenethyl isothiocyanate andN-acetylcysteine conjugates thereof.
 22. A method according to claim 21,wherein the isothiocyanate is sulforaphane.
 23. A method according toclaim 21, wherein the isothiocyanate is phenethyl isothiocyanate.
 24. Amethod according to claim 21, wherein the isothiocyanate is theN-acetylcysteine conjugate of sulforaphane.
 25. A method according toclaim 21, wherein the isothiocyanate is the N-acetylcysteine conjugateof phenethyl isothiocyanate.
 26. A method according to claim 21, whereinthe isothiocyanate is administered to the mammal as a dietarysupplement.
 27. A method according to claim 21, wherein the mammal is ahuman.
 28. A method according to claim 27, wherein the isothiocyanate isadministered to the human in a dosage of between about 0.5 and 12 mg/kgbody weight per day.
 29. A method according to claim 21, wherein theisothiocyanate is administered to the mammal in a substantially purifiedform.
 30. A method according to claim 21, wherein the isothiocyanate isadministered to the mammal in a composition comprising theisothiocyanate and a pharmacologically acceptable carrier, excipient ordiluent.
 31. A method according to claim 22, wherein the sulforaphane isadministered to the mammal in a substantially purified form.
 32. Amethod according to claim 22, wherein the sulforaphane is administeredto the mammal in a composition consisting essentially of thesulforaphane and a pharmacologically acceptable carrier, excipient ordiluent.
 33. A method according to claim 22, wherein the mammal is ahuman and the sulforaphane is administered to the human in a dosage ofbetween about 0.55 and 1.1 mg/kg body weight per day.
 34. A methodaccording to claim 23, wherein the phenethyl isothiocyanate isadministered to the mammal in a substantially purified form.
 35. Amethod according to claim 23, wherein the phenethyl isothiocyanate isadministered to the mammal in a composition consisting essentially ofthe phenethyl isothiocyanate and a pharmacologically acceptable carrier,excipient or diluent.
 36. A method according to claim 23, wherein themammal is a human and the phenethyl isothiocyanate is administered tothe human in a dosage of between about 0.6 and 1.2 mg/kg body weight perday.
 37. A method according to claim 24, wherein the N-acetylcysteineconjugate of sulforaphane is administered to the mammal in asubstantially purified form.
 38. A method according to claim 24, whereinthe N-acetylcysteine conjugate of sulforaphane is administered to themammal in a composition consisting essentially of the N-acetylcysteineconjugate of sulforaphane and a pharmacologically acceptable carrier,excipient or diluent.
 39. A method according to claim 24, wherein themammal is a human and the N-acetylcysteine conjugate of sulforaphane isadministered to the human in a dosage of between about 5.5 and 11 mg/kgbody weight per day.
 40. A method according to claim 25, wherein theN-acetylcysteine conjugate of phenethyl isothiocyanate is administeredto the mammal in a substantially purified form.
 41. A method accordingto claim 25, wherein the N-acetylcysteine conjugate of phenethylisothiocyanate is administered to the mammal in a composition consistingessentially of the N-acetylcysteine conjugate of phenethylisothiocyanate and a pharmacologically acceptable carrier, excipient ordiluent.
 42. A method according to claim 25, wherein the mammal is ahuman and the N-acetylcysteine conjugate of phenethyl isothiocyanate isadministered to the human in a dosage of between about 6.0 and 12 mg/kgbody weight per day.